- 學位論文
以轉錄體學探討亞磷酸誘導番茄對疫病菌之抗性
Transcriptome analysis of neutralized phosphorous acid-induced tomato resistance against Phytophthora parasitica
摘要
以亞磷酸處理植物對卵菌引起的病害有良好的防治效果，但亞磷酸誘導植物抗病所涉機制一直仍待深入探討。本研究應用生物晶片技術分析番茄轉錄體，發現無論是亞磷酸處理或對照組植株，於疫病菌(Phytophthora parasitica)感染初期，許多基礎代謝相關基因呈現表現差異，表示這些基因表現並非因亞磷酸處理而產生變化，而是疫病菌接種所致。這些基因大部分涉及降解物質，包含醣類、脂質及胺基酸等，這樣的現象可能與細胞遇到逆境時的能量需求有關。甘油脂(glycerolipid)、丙酸(propanoate)以及醣脂(glycosphingolipid)等脂質之代謝相關基因僅於亞磷酸處理植物被疫病菌感染時才特別表現，顯示其參與植物抗病的可能性。此外，晶片分析也顯示Atg3、Atg6與Atg18等自噬作用(autophagy)相關基因於番茄植株被疫病菌感染時，表現量明顯提升。為探討自噬作用於亞磷酸誘導抗性的角色，本研究以即時定量聚合酶鏈鎖反應分析基因表現，發現因應疫病菌侵染，對照組植物之Atg3, Atg6與Atg18等基因的表現明顯提升，但在亞磷酸處理植物的變化不大。另以eGFP標定自噬體(autophagosome)關鍵蛋白Atg8-2 (馬鈴薯ATG8CL的同源基因)，發現對照組葉片被疫病菌感染時，會形成許多自噬體 ，顯示自噬作用會因應疫病菌侵染而發生，但在亞磷酸處理植物，自噬體的數量明顯減少。另外以Tobacco rattle virus (TRV)-mediated gene silencing靜默Atg3, Atg6, Atg8-2,或Atg18 (阿拉伯芥Atg18b之同源基因)的基因表現，對於植物感病度及亞磷酸誘導抗性並無顯著影響，但Atg8-4被靜默時，亞磷酸所誘導番茄的抗性明顯較差，顯示其可能參與亞磷酸誘發抗性的路徑。這些結果顯示植物與疫病菌的交互作用涉及許多代謝相關基因的差異表現，且脂質代謝的基因可能參與亞磷酸誘導抗性。另外，因應疫病菌接種，植物會大幅度啟動自噬作用，但在亞磷酸處理植物則不顯著；然而基因靜默Atg8-4會降低亞磷酸所誘導抗性，顯示自噬作用很可能參與亞磷酸誘導抗性。
並列摘要
Neutralized phosphorous acid (NPA) is widely used for the management of plant diseases caused by oomycete pathogens including Phytophthora parasitica. While it has been proposed that phosphonate, the effective component of NPA, may function through enhancing plant resistance, the detailed mechanism remains to be elucidated. To identify genes which might play key roles in NPA-induced resistance, microarray analysis was performed. The results showed that various genes are differentially expressed in NPA- and water-pretreated (as a control) tomato plants in response to infection by P. parasitica, with many of them involved in primary metabolisms, such as degradation of carbohydrates, amino acids, and fatty acids. In addition, following pathogen infection, genes involved in lipid metabolism were identified only in NPA-pretreated plants, which suggests their involvement in NPA-induced resistance. As well, autophagy-related genes including Atg3, Atg6, and Atg18 (a homolog of Arabidopsis thalianaAtg18b) were significantly upregulated in NPA-pretreated tomato plants as shown by microarray data. However, analysis by qRT-PCR indicated that these genes are upregulated only in the control but not NPA-pretreated plants. Examination by confocal microscopy of the subcellular distribution of Atg8-2-GFP, a potato Atg8CL homolog encoding a key protein of the autophagosome, demonstrated that infection by P. parasitica induced in Nicotiana benthamiana epidermal cells the formation of abundant autophagosomes, which are significantly reduced in NPA-pretreated plants. Tobacco rattle virus-mediated gene silencing of Atg3, Atg6, Atg8-2, and Atg18 did not alter tomato susceptibility towards the pathogen regardless of NPA treatment. However, downregulation of Atg8-4 did enhance disease symptom in the NPA-treated plants. These results suggest an important role of primary metabolism in plant response towards infection of P. parasitica, as well as a role of lipid metabolism and Atg8-4 in NPA-induced resistance.
參考文獻
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